Tomato pathogenic fungus detecting apparatus and detecting method using same

ABSTRACT

The present disclosure provides a simple and secure apparatus and a simple and secure method for selectively detecting a tomato pathogenic fungus. The tomato pathogenic fungus detecting apparatus according to the present disclosure is characterized by including an artificial cell wall, a test sample solution inlet provided above the artificial cell wall, and a culture solution storage part provided under the artificial cell wall, wherein a test sample solution contains a 50 mM to 70 mM buffer solution of a citrate salt in the test sample solution inlet, and the test sample solution has a pH of 5 to 5.5.

TECHNICAL FIELD

The present invention relates to a tomato pathogenic fungus detectingapparatus and a selective detecting method using the tomato pathogenicfungus detecting apparatus.

BACKGROUND ART

Phytopathogenic fungi have, as properties involving invasiveness intoplants, features of forming an appressorium on a surface of a plant forattachment, and then searching for a pore, such as a stoma tissue,through which a hypha is extended into a plant body or secreting a plantcell wall degrading enzyme (a cellulase or a pectinase) from a hypha.

Making use of these features, for example, PTL 1 discloses a method formeasuring an amount of a fungus using a microporous membrane support.NPL 1 discloses facts that a pseudohypha of Phytophthora sojae as onetype of phytopathogenic oomycete grows downward as if attempting to godeep, rather than growing horizontally and that the pseudohyphapenetrates a PET (polyethylene terephthalate) membrane having a pore of3 μm.

Focusing on this property, the inventors of the present invention havealready proposed a method for determining a phytopathogenic oomycete(PTL 2).

CITATION LIST Patent Literatures

PTL 1: Japanese Patent Application Publication No. 2005-287337

PTL 2: Japanese Patent No. 6,167,309

PTL 3: WO 2018/011835 A

Non-Patent Literatures

NPL 1: Paul F. Morris, et. al. “Chemotropic and Contact Responses ofPhytophthora sojae Hyphae to Soybean Isoflavonoids and ArtificialSubstrates”, Plant Physiol. (1998) 117: 1171-1178

NPL 2: Noboru Shirane et al., “Mineal Salt Medium for the Growth ofBotrytis cinerea in vitro”, Ann. Phytopath. Soc. Japan 53: 191-197(1987)

SUMMARY OF THE INVENTION Technical Problem

A target plant in the present invention, tomatoes are highly prone todisease caused by fungi, and pathogenic fungi that cause the disease aresaid to be dominated by three types of fungi, a tomato gray mold fungus(Botrytis cinerea, a tomato sooty mold fungus (Pseudocercosporafuligena), and a tomato leaf mold fungus (Passalora fulva). With regardto these pathogenic fungi, the gray mold fungus (Botrytis cinerea) isplurivorous and is infectious also to other plants, but the sooty moldfungus (Pseudocercospora fuligena) and the leaf mold fungus (Passalorafulva) present examples of infection only to tomatoes and are pathogenicfungi having high plant specificity. With regard to these pathogenicfungi that are said to have specificity to tomatoes, the inventors ofthe present invention have considered that it is necessary to detect thetomato pathogenic fungi in a stage where it is unclear what type offungus is present on actual tomato leaves, that is, in a stage beforepathogenesis, and have studied on this subject.

On the other hand, a pathogenic fungus selection technique using anartificial cell wall that is a basic selective fungus detectiontechnique described in PTL 2 and used by the inventors of the presentinvention probably detects not only the tomato pathogenic fungi but alsoany phytopathogenic fungi. That is, if a fungus pathogenic to anotherplant is attached to a tomato leaf, the pathogenic fungus selectiontechnique may possibly detect the fungus as a tomato pathogenic fungus.Tomato cultivation is mostly performed not by seeds but by seedlings,and a possibility cannot be ruled out of attaching a phytopathogenicfungus other than the tomato pathogenic fungi to a tomato seedling in anursery garden, due to mixed cultivation with other plants and sharingof tools among a plurality of plants in a same facility. Similarly tothe nursery garden described above, there is a possibility of attachinga fungus pathogenic to a plant other than tomatoes to a tomato seedlingalso in an actual cultivation site and a cultivation facility such as avinyl greenhouse. If such attachment is left untreated, thephytopathogenic fungus other than the tomato pathogenic fungi possiblyleads to presentation of a false positive in the pathogenic fungusselection technique using an artificial cell wall, to sometimes causesevere inconvenience in cultivation, such as useless chemicalapplication or renewal of seedlings.

As a result of a research and investigation on this possibility ofgenerating a false positive, the inventors of the present invention haveactually encountered fungi that are other than the tomato pathogenicfungi and that lead to presentation of a false positive in a studyingdetecting method using an artificial cell wall. The fungi are four typesof fungi, a Biscogniauxia genus fungus, a Penicillium genus fungus, aPhoma genus fungus, and a Trichoderma genus fungus, and a study on amethod that does not detect these fungi has been required.

The present invention has been made in view of such actualcircumstances, and an object of the present invention is to provide anapparatus and a method for selectively detecting a tomato pathogenicfungus.

Solution to Problem

As a result of an earnest study, the inventors of the present inventionand others have found that a detecting apparatus configured as below cansolve the above problem and further conducted the study based on thefinding to complete the present invention.

That is, a tomato pathogenic fungus detecting apparatus related to oneaspect of the present invention is characterized by including anartificial cell wall, a test sample solution inlet provided above theartificial cell wall, and a culture solution storage part provided underthe artificial cell wall, wherein a test sample solution contains a 50mM to 70 mM buffer solution of a citrate salt in the test samplesolution inlet, and the test sample solution has a pH of 5 to 5.5.

Advantageous Effects of Invention

The present invention is capable of providing an apparatus and a methodthat are capable of selectively detecting a tomato pathogenic fungussimply and safely. The present invention enables presence of a tomatopathogenic fungus to be detected in a stage before pathogenesis causedby the fungus and enables presentation of a false positive attributed toa phytopathogenic fungus other than tomato pathogenic fungi to beavoided in the detection, so that the present invention is very usefulfor industrial use.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic sectional view illustrating one example of adetecting apparatus of the present embodiment.

FIG. 2 shows a schematic sectional view illustrating one example of anartificial cell wall included in the detecting apparatus of the presentembodiment.

FIG. 3 shows a schematic sectional view illustrating one example of thedetecting apparatus of the present embodiment.

FIG. 4 shows a micrograph of a back surface of an artificial cell wallillustrating a state where a tomato gray mold fungus (Botrytis cinerea)has penetrated the artificial cell wall in Example 1.

FIG. 5 shows a graph illustrating results of Comparative Example 1.

FIG. 6 shows a graph illustrating results of Example 1.

FIG. 7 shows a graph illustrating results of Comparative Example 2.

FIG. 8 shows a graph illustrating results of Comparative Example 3.

FIG. 9 shows a graph illustrating results of Comparative Example 4.

FIG. 10 shows a graph illustrating results of Comparative Example 5.

FIG. 11 shows a graph illustrating results of Comparative Example 6.

DESCRIPTION OF EMBODIMENT

Hereinafter, an embodiment according to the present invention isspecifically described. The present invention, however, is not limitedto this embodiment.

Tomato pathogenic fungus detecting apparatus 1 according to the presentembodiment is characterized by including, as illustrated in FIG. 1,artificial cell wall 2, test sample solution inlet 3 provided aboveartificial cell wall 2, and culture solution storage part 4 providedunder artificial cell wall 2. Test sample solution 5 contains a 50 mM to70 mM buffer solution of a citrate salt in test sample solution inlet 3.Test sample solution 5 has a pH of 5 to 5.5.

Test sample solution inlet 3 is a vessel into which test sample solution5 is charged, and the vessel desirably includes a flange on an upper endof the vessel. A bottom surface of test sample solution inlet 3 isformed of artificial cell wall 2.

Artificial cell wall 2 preferably includes, as illustrated in FIG. 2, atleast substrate 21 having through hole 22, and cellulose membrane 23provided on one surface of substrate 21. Use of such an artificial cellwall further facilitates selective detection of a targeted tomatopathogenic fungus.

Through hole 22 penetrates from a front-end surface to a back-endsurface of substrate 21, and the through hole preferably has a holediameter of 2 μm to 7 μm (sectional area of 4.5 μm² to 38.5 μm²). Thethrough hole having a hole diameter in the above range enables a targetpathogenic fungus to be selectively detected more securely.

Further, in order to selectively detect a target pathogenic fungus moresecurely, a thickness of cellulose membrane 23 is also preferablyadjusted. Specifically, cellulose membrane 23 preferably has a thicknessof 0.5 μm to 2 μm.

It is considered that in artificial cell wall 2 of the presentembodiment, adjusting the hole diameter of through hole 22 in substrate21 and the membrane thickness of cellulose membrane 23 in the aboverange does not allow many of tomato non-pathogenic fungi to penetratecellulose membrane 23, so that a part of the tomato non-pathogenic fungican be excluded in this stage. On the other hand, a tomato pathogenicfungus targeted in the present embodiment selectively appears on theback surface of the substrate.

A thickness of substrate 21 is not particularly limited, but ispreferably about 5 μm to 150 μm as one example.

As illustrated in FIG. 1, test sample solution 5 is supplied into testsample solution inlet 3. Thus, if test sample solution 5 contains atomato pathogenic fungus, the tomato pathogenic fungus comes to bepresent on the front-end surface of substrate 21.

In the present embodiment, test sample solution 5 is mainly a solution(fungus collection solution) containing a fungus attached to a tomatoleaf, and is not particularly limited as long as the test samplesolution is a liquid probably containing a target pathogenic fungus. Thetest sample solution is, for example, a liquid having been used to washa tomato leaf or a liquid in which a tomato leaf has been immersed.

In the present embodiment, it is important that test sample solution 5has a pH of 5 to 5.5 and test sample solution 5 contains a 50 mM to 70mM buffer solution of a citrate salt. These configurations enableobstructive fungi (tomato non-pathogenic fungi) that lead to a falsepositive in pathogenic fungus detection to be excluded and thus enable atarget tomato pathogenic fungus to be selectively detected.

Test sample solution 5 having a pH of less than 5 or more than 5.5 maypossibly make it impossible to completely exclude the tomatonon-pathogenic fungi that obstruct the tomato pathogenic fungusdetection. The test sample solution that contains the buffer solutionhaving a concentration of the citrate salt of less than 50 mM maypossibly make it impossible to completely exclude the tomatonon-pathogenic fungi that obstruct the tomato pathogenic fungusdetection. On the other hand, the test sample solution that contains thebuffer solution having a concentration of the citrate salt of more than70 mM may possibly also exclude a part of targeted tomato pathogenicfungi.

The citrate salt is not particularly limited, but is preferably amonovalent citrate salt, and is preferably sodium citrate, potassiumcitrate, or the like more specifically.

Further, test sample solution 5 normally preferably has an EC (electricconductivity) of about 7 mS/cm to 15 mS/cm.

The tomato pathogenic fungus targeted by the detecting apparatus of thepresent embodiment is preferably at least one selected from a tomatogray mold fungus (Botrytis cinerea), a tomato sooty mold fungus(Pseudocercospora fuligena), or a tomato leaf mold fungus (Passalorafulva).

The detecting apparatus of the present embodiment preferably does notdetect fungi that are sometimes present on tomato leaves but are tomatonon-pathogenic fungi, e.g., a Biscogniauxia genus fungus, a Penicilliumgenus fungus, a Phoma genus fungus, and a Trichoderma genus fungus. Morespecifically, the tomato non-pathogenic fungi are Biscogniauxiamaritima, Penicillium olsonii, Phoma multirostrata, and Trichodermaasperellum.

In the present specification, the term “tomato pathogenic” means beingpathogenic to tomatoes. The term “tomato non-pathogenic” means beingnon-pathogenic to tomatoes. A fungus that is pathogenic but is notpathogenic to tomatoes is “tomato non-phytopathogenic”. In other words,a fungus that does not adversely affect tomatoes is “tomatonon-pathogenic”. The prefix “non-” included in the term “tomatonon-pathogenic” does not modify the “tomato”, but modifies the“pathogenic”.

In the detecting apparatus of the present embodiment, a culture solutionis put in culture solution storage part 4 provided under artificial cellwall 2. The culture solution is not particularly limited as long as theculture solution is capable of culturing a fungus, and a general culturemedium or culture solution is usable. For example, general culture mediafor culturing a fungus, i.e., a potato dextrose culture medium,Sabouraud dextrose culture medium, and the like are usable. In order toaccelerate the culture of a fungus, a culture solution may be added notonly to culture solution storage part 4 but also to test sample solution5.

The detecting apparatus of the present embodiment detects presence orabsence of a tomato pathogenic fungus in a sample by observing, after alapse of a certain culture period, whether or not the tomato pathogenicfungus has appeared on the back surface of cellulose membrane 23 ofartificial cell wall 2. An observation means is not particularlylimited, and optical observation can be conducted with microscope 6 bydisposing microscope 6 under artificial cell wall 2 as illustrated inFIG. 3.

The culture period of a fungus is not particularly limited and ispreferably not less than 72 hours. A culture temperature is preferablyset at about 20° C. to 28° C.

The present invention further encompasses a tomato pathogenic fungusdetecting method including selectively detecting a tomato pathogenicfungus using the detecting apparatus described above.

The tomato pathogenic fungus detecting method of the present embodimentis not particularly limited in terms of steps other than using thedetecting apparatus described above, and includes the steps of, forexample: charging a test sample solution into test sample solution inlet3 of the detecting apparatus; leaving the test sample solution to standstill in the detecting apparatus (culturing); observing a back surfaceof artificial cell wall 2 (cellulose membrane 23) in the detectingapparatus after the leaving; and determining that the test samplesolution contains a tomato pathogenic fungus when the fungus is observedon the back surface of cellulose membrane 23.

The present specification discloses various forms of techniques asdescribed above, from among which main techniques are summarized asfollows.

A tomato pathogenic fungus detecting apparatus according to one aspectof the present invention is characterized by including an artificialcell wall, a test sample solution inlet provided above the artificialcell wall, and a culture solution storage part provided under theartificial cell wall, wherein a test sample solution contains a 50 mM to70 mM buffer solution of a citrate salt in the test sample solutioninlet, and the test sample solution has a pH of 5 to 5.5.

These configurations enable provision of an apparatus and a method thatare capable of selectively detecting a tomato pathogenic fungus simplyand safely.

In the detecting apparatus, it is preferable that the artificial cellwall includes at least a substrate that has a through hole with a holediameter of 2 μm to 7 μm and has a thickness of 5 μm to 150 μm, and acellulose membrane that is provided on one surface of the substrate andhas a thickness of 0.5 μm to 2 μm. These configurations are consideredto enable the effects described above to be more securely obtained.

In the detecting apparatus, the citrate salt is preferably at least oneselected from sodium citrate or potassium citrate. This setting isconsidered to enable the effects described above to be more securelyobtained.

In the detecting apparatus, a tomato pathogenic fungus to be a detectiontarget is preferably at least one selected from a tomato gray moldfungus (Botrytis cinerea), a tomato sooty mold fungus (Pseudocercosporafuligena), or a tomato leaf mold fungus (Passalora fulva). This settingis considered to enable the effects described above to be moreexhibited.

The detecting apparatus preferably does not detect fungi that aresometimes present on tomato leaves but are tomato non-pathogenic fungi,namely a Biscogniauxia genus fungus, a Penicillium genus fungus, a Phomagenus fungus, and a Trichoderma genus fungus. This setting is consideredto enable the effects described above to be more exhibited.

The tomato non-pathogenic fungi are more preferably Biscogniauxiamaritima, Penicillium olsonii, Phoma multirostrata, and Trichodermaasperellum.

A tomato pathogenic fungus detecting method according to another aspectof the present invention is characterized by selectively detecting atomato pathogenic fungus using the detecting apparatus.

Hereinafter, the present invention is described further specifically byway of an example. A scope of the present invention, however, is notlimited to this example.

EXAMPLE Preparation of Fungi Culture of Botrytis cinerea

Botrytis cinerea, which is one of tomato pathogenic fungi and apathogenic fungus of tomato gray mold disease was inoculated into apotato dextrose agar culture medium (Difco™ Potato Dextrose Agar). Next,the culture medium was left to stand still at a temperature of 25degrees Celsius for one week. Botrytis cinerea was given by associateprofessor Shimizu belonging to Faculty of Applied Biological Sciences,Gifu University. Thereafter, the Botrytis cinerea-cultured potatodextrose agar culture medium in which hyphae sufficiently grew was leftunder irradiation with black light for not less than four days and leftin a room-temperature environment for not less than two weeks to promotespore formation. Several ml of sterile pure water was dropped to thetreated Botrytis cinerea-cultured potato dextrose agar culture medium,and surfaces of the hyphae were rubbed with a platinum loop, an inkbrush, or the like to give a crushed hypha and spore mixed suspension.

Culture of Pseudocercospora fuligena

Pseudocercospora fuligena, which is one of tomato pathogenic fungi and apathogenic fungus of tomato sooty mold disease was inoculated into apotato dextrose agar culture medium. Next, the culture medium was leftto stand still at a temperature of 28 degrees Celsius for one week.Pseudocercospora fuligena was gotten from The Genetic Resources Center,NARO (the National Agriculture and Food Research Organization) (MAFF No.306728). Thereafter, hyphae of Pseudocercospora fuligena weretransplanted from the potato dextrose agar culture medium to a burdockpowder agar culture medium, and further left to stand still at atemperature of 28 degrees Celsius for one to two weeks. Aftersufficiently growing again, the hyphae was subjected to mechanicalstress such as rubbing surfaces of the hyphae with a platinum loop, anink brush, or the like, thereafter left under irradiation with blacklight for not less than four days, and then left in a room-temperatureenvironment for not less than two weeks to promote spore formationagain. Several ml of sterile pure water was dropped to the treatedPseudocercospora fuligena-cultured burdock powder agar culture medium,and surfaces of the hyphae were rubbed with a platinum loop, an inkbrush, or the like to give a crushed hypha and spore mixed suspension.

Culture of Passalora fulva

Passalora fulva, which is one of tomato pathogenic fungi and apathogenic fungus of tomato leaf mold disease was inoculated into apotato dextrose agar culture medium. Next, the culture medium was leftto stand still at a temperature of 23 degrees Celsius for one to twoweeks. Passalora fulva was gotten from The Genetic Resources Center,NARO (the National Agriculture and Food Research Organization) (MAFF No.726744). Thereafter, several ml of sterile pure water was dropped to thePassalora fulva-cultured potato dextrose agar culture medium in whichhyphae sufficiently grew, and surfaces of the hyphae were rubbed with aplatinum loop, an ink brush, or the like to give a crushed hypha andspore mixed suspension.

Culture of Biscogniauxia maritima, Penicillium olsonii, Phomamultirostrata, and Trichoderma asperellum

Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, andTrichoderma asperellum that were not tomato pathogenic fungi but werepresent on tomato leaves were collected from the tomato leaves,separated, and then inoculated into a potato dextrose agar culturemedium. Separation sources, tomatoes were collected from a plurality oflocations. A separation method was as follows. Collected several tomatoleaves were charged into a clear resin vessel or a resin bag togetherwith a fungus collection solution that consists of saline containing0.1% of surfactant Tween 80 (SIGMA-ALDRICH), stirred for one minute totransfer fungi attached to the leaves to the fungus collection solution.The fungus collection solution was diluted and applied to a potatodextrose agar culture medium containing 100 mg/L of streptomycin sulfate(Wako Pure Chemical Industries, Ltd.) by a plate agar smear method.Then, fungi that emerged in culture at 25 degrees Celsius for severaldays were separated from a fungus colony. Identification of the fungiwas commissioned to Japan Food Research Laboratories (generalincorporated foundation), Tama Laboratory. After the isolation,Biscogniauxia maritima, Penicillium olsonii, Phoma multirostrata, andTrichoderma asperellum that were inoculated into potato dextrose agarculture media were left to stand still at a temperature of 25 degreesCelsius for one week. Thereafter, several ml of sterile pure water wasdropped to the potato dextrose agar culture media for culturing thesefour types of fungi in which hyphae sufficiently grew or spores weresufficiently formed, and surfaces of the hyphae were rubbed with aplatinum loop, an ink brush, or the like to give a crushed hypha andspore mixed suspension.

Preparation of Artificial Cell Wall

The artificial cell wall in the detecting apparatus was prepared asfollows.

First, cellulose (SIGMA-ALDRICH, trade name: Avicel PH-101) wasdissolved in an ionic liquid to prepare a cellulose solution having aconcentration of 1%. The ionic liquid was 1-Butyl-3-methyl imidazoliumchloride (manufactured by SIGMA-ALDRICH). The cellulose solution washeated to 60 degrees Celsius and next applied to a back surface of avessel (Millipore, trade name: Millicell PISP 12R 48) including apolyethylene terephthalate film as a bottom surface by spin coating for30 seconds at a rotation rate of 2000 rpm. The polyethyleneterephthalate film functioned as substrate 21 of the artificial cellwall in FIG. 2 and randomly had a plurality of through holes with adiameter of 3 μm. Thus, a cellulose membrane having a thickness of 0.5micrometers was formed on a back-end surface of the polyethyleneterephthalate film.

The vessel including the cellulose membrane formed on the back surfaceof the polyethylene terephthalate film as the bottom surface was left tostand still in ethanol for 12 hours at room temperature. Thus,1-Butyl-3-methyl imidazolium chloride was replaced with ethanol andremoved, and then the vessel was dried in a vacuum desiccator at theend. Thus, the artificial cell wall was obtained that was tested in thepresent example and comparative examples.

Preparation of Tomato Pathogenic Fungus Detecting Apparatus

The vessel that was formed into the artificial cell wall and includedthe cellulose membrane on the back surface of the polyethyleneterephthalate film (substrate) as the bottom surface was put on aculture medium vessel (culture solution storage part) to form a tomatopathogenic fungus detecting apparatus. As the culture medium vessel, a24-well flat bottom culture plate (Corning incorporated, trade name: 24Well Cell Culture Cluster Flat Bottom) was used, and a space between theculture medium vessel and the artificial cell wall-forming vessel wasfilled with 600 μL of a liquid culture medium (culture solution) so thatthe back surface of the artificial cell wall-forming vessel was incontact with the liquid culture medium. As the liquid culture medium, adiluted potato dextrose liquid culture medium (Difco™ Potato DextroseBroth 2.4 g/L aqueous solution) was used.

Example 1

The crushed hypha and spore mixed suspensions respectively containing200 pieces of hyphae and spores of Botrytis cinerea, Pseudocercosporafuligena, Passalora fulva, Biscogniauxia maritima, Penicillium olsonii,Phoma multirostrata, and Trichoderma asperellum were separately addedinto the artificial cell wall-forming vessel, and a sodium citratebuffer solution was added to the vessel in the example so that a totalvolume of each of the resultant crushed hypha and spore mixedsuspensions and the sodium citrate buffer solution became 200 μL. Thus,test sample solutions were obtained. The sodium citrate buffer solutionwas prepared and added so that a concentration of the sodium citratebuffer solution contained in the test sample solution became 60 mM whenthe sodium citrate buffer solution was mixed with the crushed hypha andspore mixed suspension to give a volume of 200 μL. The sodium citratebuffer solution added into the artificial cell wall-forming vessel had apH of 5.5 and an EC of 13 mS/cm.

Then, the test sample solutions respectively containing the seven typesof fungi were disposed in the prepared detecting apparatus, which wasleft to stand still at a temperature of 25 degrees Celsius and subjectedto observation at intervals of 24 hours. A number of hyphae thatpenetrated the artificial cell wall and observed on the back surface ofthe artificial cell wall was counted every 24 hours by visual inspectionvia an optical microscope. FIG. 4 shows one example (tomato gray moldfungus (Botrytis cinerea)) of observation photographs captured with theoptical microscope.

Comparative Example 1

A test was performed similarly to in Example 1 except that sterilepurified water was used in place of the sodium citrate buffer solution.

Consideration

FIG. 5 shows results of Comparative Example 1 and FIG. 6 shows resultsof Example 1.

FIG. 5 shows that hyphae having penetrated the artificial cell wall wereobserved earlier in the four types of tomato non-pathogenic fungi thatwere sometimes present on tomato leaves but were to be excluded from thedetection, namely Biscogniauxia maritima, Penicillium olsonii, Phomamultirostrata, and Trichoderma asperellum, than in the tomato pathogenicfungi to be detected, namely Pseudocercospora fuligena and Passalorafulva. Thus, the tomato pathogenic fungi could not be selectivelydetected in the present comparative example.

In contrast, FIG. 6 representing the results of the example shows thathyphae having penetrated the artificial cell wall were, at 72 h,observed earlier in the tomato pathogenic fungi, Botrytis cinerea,Pseudocercospora fuligena, and Passalora fulva than in the four types oftomato non-pathogenic fungi that were sometimes present on tomato leavesbut were to be excluded from the detection, namely Biscogniauxiamaritima, Penicillium olsonii, Phoma multirostrata, and Trichodermaasperellum. Thus, it was confirmed that the tomato pathogenic fungicould be selectively detected in Example 1.

Comparative Example 2

A test was performed similarly to in Example 1 except that a sodiumcitrate buffer solution having the same concentration (60 mM) as in thetest sample solution was charged also into the culture medium of thedetecting apparatus and the culture solution was prepared to also have apH of 5.5. FIG. 7 shows results.

As is clear from FIG. 7, any of the fungi neither penetrated theartificial cell wall nor grew in Comparative Example 2.

Comparative Example 3

A test was performed similarly to in Example 1 except that the pH of thetest sample solution was changed to 4.5. FIG. 8 shows results of thenumber of invading hyphae after 72 hours of the culture in ComparativeExample 3.

As is clear from FIG. 8, a part of the obstructive fungi (tomatonon-pathogenic fungi) penetrated the artificial cell wall, and theobstructive fungi could not be completely excluded in ComparativeExample 3.

Comparative Example 4

A test was performed similarly to in Example 1 except that the pH of thetest sample solution was changed to 6. FIG. 9 shows results of thenumber of invading hyphae after 72 hours of the culture in ComparativeExample 4.

As is clear from FIG. 9, a part of the obstructive fungi (tomatonon-pathogenic fungi) penetrated the artificial cell wall, and theobstructive fungi could not be completely excluded also in ComparativeExample 4.

The results of Comparative Examples 3 and 4 above indicated that the pHof the test sample solution is one of important factors for selectivelydetecting a tomato pathogenic fungus.

Comparative Example 5

A test was performed similarly to in Example 1 except that the pH of thetest sample solution was set at 5 and the concentration of the sodiumcitrate buffer solution contained in the test sample solution was set at100 mM. FIG. 10 shows results of the number of invading hyphae after 72hours of the culture in Comparative Example 5.

The results in FIG. 10 made it understandable that Comparative Example 4not only excluded the obstructive fungi (tomato non-pathogenic fungi)but also a part of the target tomato pathogenic fungi.

Comparative Example 6

A test was performed similarly to in Example 1 except that the pH of thetest sample solution was set at 5 and the concentration of the sodiumcitrate buffer solution contained in the test sample solution was set at40 mM. FIG. 11 shows results of the number of invading hyphae after 72hours of the culture in Comparative Example 6.

As is clear from FIG. 11, a part of the obstructive fungi (tomatonon-pathogenic fungi) penetrated the artificial cell wall, and theobstructive fungi could not be completely excluded in ComparativeExample 6.

The results of Comparative Examples 5 and 6 above indicated that theconcentration of the sodium citrate buffer solution contained in thetest sample solution is one of important factors for selectivelydetecting a tomato pathogenic fungus.

INDUSTRIAL APPLICABILITY

A tomato pathogenic fungus detecting apparatus of the present disclosureis capable of selectively detecting a target tomato pathogenic funguswhile excluding a tomato non-pathogenic fungus leading to a falsepositive. Therefore, the detecting apparatus of the present disclosurecan be suitably utilized for removing a tomato pathogenic fungus thatadversely affects tomatoes or for other purposes in technical fieldssuch as agriculture involving tomatoes.

REFERENCE SIGNS LIST

1 detecting apparatus

2 artificial cell wall

3 test sample solution inlet

4 culture solution storage part

5 test sample solution

6 microscope

21 substrate

22 through hole

23 cellulose membrane

1. A tomato pathogenic fungus detecting apparatus comprising: an artificial cell wall; a test sample solution inlet provided above the artificial cell wall; and a culture solution storage part provided under the artificial cell wall, wherein a test sample solution contains a 50 mM to 70 mM buffer solution of a citrate salt in the test sample solution inlet; and the test sample solution has a pH of 5 to 5.5.
 2. The tomato pathogenic fungus detecting apparatus according to claim 1, wherein the artificial cell wall includes a substrate that has a through hole with a hole diameter of 2 μm to 7 μm and has a thickness of 5 μm to 150 μm, and a cellulose membrane that is provided on one surface of the substrate and has a thickness of 0.5 μm to 2 μm.
 3. The tomato pathogenic fungus detecting apparatus according to claim 1, wherein the citrate salt is at least one selected from the group consisting of sodium citrate and potassium citrate.
 4. The tomato pathogenic fungus detecting apparatus according to claim 1, wherein a tomato pathogenic fungus to be a detection target is at least one selected from the group consisting of tomato gray mold fungus Botrytis cinerea, tomato sooty mold fungus Pseudocercospora fuligena, and a tomato leaf mold fungus Passalora fulva.
 5. The tomato pathogenic fungus detecting apparatus according to claim 1, wherein the tomato pathogenic fungus detecting apparatus does not detect a Biscogniauxia genus fungus, a Penicillium genus fungus, a Phoma genus fungus, and a Trichoderma genus fungus, these four of which are tomato non-pathogenic fungi.
 6. The tomato pathogenic fungus detecting apparatus according to claim 5, wherein the Biscogniauxia genus fungus is Biscogniauxia maritima, the Penicillium genus fungus is Penicillium olsonii, the Phoma genus fungus is Phoma multirostrata, and the Trichoderma genus fungus is Trichoderma asperellum.
 7. A tomato pathogenic fungus detecting method comprising selectively detecting a tomato pathogenic fungus using the detecting apparatus according to claim
 1. 